Reverse transcriptase-dependent viruses (RTDV) are those viruses whose genomes encode and transcribe an enzyme known as reverse transcriptase [ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase or RDDP]. Illustrative of such viruses are the retroviruses or RNA tumor viruses, e.g., human T-lymphotropic viruses (HTLV), human immunodeficiency viruses (HIV), feline leukemia virus (FeLV), Moloney murine leukemia virus (Mo-MuLV), and the like, plant viruses, e.g., cauliflower mosaic virus (a double stranded DNA virus that encodes an enzyme which, when expressed in vitro, also has RDDP activity substantially the same as that of Mo-MuLV) and the like, hepatitis B virus, and others. See, for example, Varmus (1983), Nature, 304:116-117, Varmus et al., "Replication of Retroviruses," in RNA Tumor Viruses, Weiss et al., eds., Cold Spring Harbor Laboratories, p 75-134 (1985) and Toh et al. (1983) Nature, 305:827-829.
Synthesis of DNA complementary to viral RNA is thought to be required for both retroviral integration into host DNA and for the generation of new virions. For this reason, the HIV-encoded reverse transcriptase is a logical target for the development of agent for the treatment of patients with the acquired immunodeficiency syndrome [De Clercq et al. (1986) J. Med. Chem., 29:1561-1569], and with other diseases of retroviral origin.
Mitsuya et al. (1985) Proc. Natl. Acad. Sci. USA, 82:7006-7100 reported 3'-azido-3'-deoxythymidine (AZT) blocked the replication of HIV in cultured human T lymphoblasts, and inhibited the cytopathic effects of the virus. AZT was presumably phosphorylated by the T cells and converted to the 5'-triphosphate derivative. That derivative was reported by those authors to be an inhibitor of HIV reverse transcriptase activity.
Yarchoan et al. (1986) Lancet, i:575-580, administered AZT to patients with AIDS or AIDS-related disease complexes. The drug was reportedly well tolerated and crossed the blood/brain barrier.
Recently, Mitsuya et al. (1986) Proc. Natl. Acad. Sci. USA, 83:1911-1915 reported that the 2',3'-dideoxynucleoside derivatives of adenosine, guanosine, inosine, cytidine and thymidine also inhibited the infectivity and cytopathic effect of HIV in vitro at concentrations from 10-20 fold less than those that blocked the proliferation of uninfected T cells. These compounds were reported to be relatively non-toxic towards host T cells; a surprising finding. The adenosine and cytidine derivatives were reported to be more potent than the guanosine and inosine derivatives.
The 2',3'-dideoxynucleosides are phosphorylated at the 5'-position in T cells to form the 5'-nucleotide triphosphate derivatives. Those derivatives are well known to be substrates for reverse transcriptase molecules. Ono et al. (1986) Biochem. Biophys. Res. Comm., 2:498-507.
Those 2',3'-dideoxynucleoside 5'-triphosphates are also utilized by mammalian DNA polymerases beta and gamma. Waquar et al., (1984) J. Cell. Physiol., 121:402-408. They are, however, poor substrates for DNA polymerase-alpha, the main enzyme responsible for both repair and replicative DNA synthesis in human lymphocytes. In part, these properties may explain the selective anti-HIV activity of the 2',3'-dideoxy-nucelosides.
It is presumed that AZT 5'-triphosphate and 2',3'-dideoxy-5'-triphosphate derivatives are incorporated into a growing RNA or DNA chain. However, lacking a 3'-hydroxyl group that is necessary to form a 3',5'-phosphodiester group, the growing RNA or DNA (polynucleotide) terminates, and thereby, presumably, inhibits HIV replication and infection.
The antiviral activity of AZT probably requires the phosphorylation of the nuceloside by thymidine kinase. This cell cycle-dependent enzyme has very low activity in human peripheral blood lymphocytes, unless the cells are stimulated to divide by a mitogen or antigen. Pegoraro et al. (1971) Exp. Cell Res., 66:283-290. For that reason, it is unlikely that substantial amounts of 3'-azido-3'-deoxythymidine 5'-phosphate accumulates in normal human peripheral blood T cells.
Most in vitro assays for HIV infection of normal T cells require lymphocyte activation by an antigen or mitogen. Burre-Sinoussi et al. (1983) Science, 220:868-871; Povovic et al. (1984) Science 224:497-500; Levy et al. (1984) Science, 225:840-842; Klatzmann et al. Science, 225:59-63; and Dalgleish et al. (1984) Nature, 312:763-766. Although such activation may be required for before viral protein can be detected due to the relatively low in vitro pathogenicity of the virus, it is thought that at least some normal peripheral blood T lymphocytes are infected by the virus since those cells express the T4 (CD4) viral receptor. Thus, AZT and other anti-viral nucleoside substrates for thymidine kinase may not totally prevent the spread of HIV infection to the residual normal T lymphocytes that circulate in infected patients.
Recent reports indicate that HIV infects macrophages and monocytes in addition to T cells. Levy et al. (1985) Virology, 147:441-448; Gartner et al (1986) Science, 233:215-219; and Wiley et al. (1986) Proc. Natl. Acad. Sci. USA, 83:7089-7093. Monocytes and macrophages have minimal or no thymidine kinase activity, although such cells do possess deoxycytidine kinase activity [Carson et al. (1977) Proc. Natl. Acad. Sci. USA, 74:5677-5681] that can phosphorylate 2',3'-dideoxycytidine (ddC). Monocytes and macrophages also appear to lack deoxycytidine kinase activity. Chan et al. (1982) J. Cell Physiol., 111:28-32.
Thus, in normal T cells, one would expect dideoxycytidine to be more effective than AZT since the former nucleoside can be phosphorylated by intracellular enzymes whereas the latter nucleoside cannot. Neither nucleoside can be expected to be phosphorylated by monocytes and macrophages. It is thought that the lack of intracellular phosphorylation and subsequent incorporation and chain termination in monocytes and macrophages contributes to the failure of nucleosides like AZT to eradicate HIV from the patient's blood.
Preliminary studies by the present inventors and their co-workers indicate that human monocyte derived macrophages (MDM) exhibit about one-tenth to about one-fourth the nucleoside kinase activity of CEM T lymphoblasts toward uridine, deoxycytidine and thymidine, and about two-thirds the adenosine kinase activity of CEM cells. In addition, that adenosine kinase activity of MDM cells is at least about 10-fold higher than any of the other kinase activities. Those studies also indicated relatively low levels of nucleoside phosphorylation using AZT, ddC and 2',3'-dideoxyadenosine (ddA) in intact CEM T lymphoblasts and still lower levels with the MDM.
The ability of AZT, ddC and ddA to inhibit synthesis of the p24 (gag) antigen of HIV in CEM and MDM cells was also examined. For CEM cells, the results for all three compounds were similar to those discussed in Mitsuya et al. (1987) Nature, 325:773-778 with ddC providing the most inhibitory effect at the lowest concentration, followed by AZT, followed by ddA in a 3-day assay. Using the same concentrations (0.1-100 uM) in a similar 3-day assay, none of those compounds provided any inhibition of p24 (gag) production from MDM cells.
The above results explain in part the observations made in clinical trials with AZT. Those results, in part, have shown that treatment of patients with AIDS or AIDS-related complex with AZT has resulted in elevation of CD4 (T4) peripheral blood cell counts, restoration of cutaneous delayed hypersensitivity, and reduction of the rate of opportunistic infections and death; results that can be related to the effect of AZT on T cells.
However, AZT had no effect on virus isolation rates from peripheral blood cells. That result suggests that a subset of infected cells persists that represents a reservoir of continuing viral replication, and with the above work with MDM cells, indicates that macrophages constitute at least a portion of that in vivo reservoir.
That AZT and ddC were not effective in the monocyte assay was not surprising in view of the before-mentioned relatively low kinase activity for the related 2'-deoxy nucleosides found in MDM cells. The similar finding as to ddA is more puzzling in view of the much greater adenosine kinase activity of MDM cells.
Although MDM cells produced lower amounts of dideoxynucleoside 5'-triphosphates than did T lymphoblasts over a four hour time period, the magnitude of that difference cannot account entirely for the failure of these nucleosides having potent effects in T cells to inhibit HIV replication in MDM cells. Other factors such as nucleotide pools, the initial rate of nucleotide formation compared to the time of reverse transcription, and adenine deaminase activity may also influence the anti-HIV activity of those compounds.
As noted before, 2',3'-dideoxyadenosine (ddA) inhibits in vitro infectivity and cytopathatic effects of HIV. Mitsuya et al. (1986) Proc. Natl. Acad. Sci. USA, 83:1911-1915. However, ddA is a known substrate for adenosine deaminase (also known as adenosine aminohydrolase, EC 3.5.4.4), which converts the compound to 2',3'-dideoxyinosine (ddI). Frederiksen (1966) Arch. Biocyem. Biophys., 113:383-388. Adenosine deaminase levels in the blood of AIDS patients are relatively high compared to normal persons. Thus, in vivo, ddA would be expected to have little effect on HIV due to the action of endogenous adenine deaminase.
On the other hand, several 2-substituted adenosine derivatives have been reported not to be deaminated by adenosine deaminase. For example, Coddington (1965) Biochim. Biophys Acta, 99:442-451 reported that deoxyadenosine-1-N-oxide, as well as 2-hydroxy, 2-methyl, 2-chloro, 2-acetamido and 2-methylthio adenosines were neither substrates nor inhibitors for adenosine deaminase. Montgomery, in Nucleosides, Nucleotides, and Their Biological Applications, Rideout et al eds., Academic Press, New York, p 19 (1983) provides a table of comparative Km and Vmax data for the deamination of adenosine, 2-halo-adenosines, 2-halo-deoxyadenosines and 2-fluoro-arabinoadenosine; that also indicate that those 2-halo, adenosines derivatives are poor substrates for the enzyme relative to adenine itself. Stoeckler et al. (1982) Biochem. Pharm., 31:1723-1728 reported that the 2'-deoxy-2'-azidoribosyl and arabinosyl adenine derivatives were substrates for human erythrocytic adenosine deaminase, whereas work of others indicated 2-fluoroadenosine to have negligible activity with adenosine deaminase.
Both dividing and resting (normal) human T lymphocytes contain especially high levels of deoxycytidine kinase, a nucleoside phosphorylating enzyme for which both deoxyguanosine and deoxyadenosine are alternative substrates. Carson et al. (1977) Proc. Natl. Acad. Sci. USA, 74:5677-5681. Deoxyadenosine is also a substrate for adenosine kinase [Hershfield et al. (1982) J. Biol. Chem., 257:6380-6386], an enzyme also present in T lymphocytes. Additional substrates for adenosine kinase including adenosine-1-N-oxide, arabinofuranosyladenine, xylofuranosyladenine, 2'-amino-2',3'-dideoxyadenosine, 2-fluoroadenosine and 3-deoxyadenosine are reported by Lindberg et al., (1967) J. Biol. Chem., 242:350-356. Lindberg et al. also reported that a number of compounds including 2'-deoxyadenosine-1-N-oxide, 3'-deoxyadenosine-1-N-oxide and 2',3'-dideoxyadenosine were neither substrates nor inhibitors for the enzyme.
T lymphocytes also contain relatively low levels of cytoplasmic 5'-nucleotidase, an enzyme that returns 5'-nucleotides to their respective nucleoside forms. As a result of the relatively high 5'-phosphorylating activity and low dephosphorylating activity of T lymphocytes, as well as the difficulty of ionically charged compounds such as nucleotides in traversing cellular membranes, human T lymphocytes tend to sequester deoxyadenosine 5'-triphosphate and deoxyguanosine 5'-triphosphate when exposed to relatively low external concentrations of the respective nucleosides.
Norman human T lymphocytes form little thymidine 5'-triphosphate when exposed to exogenous thymidine. In contrast, normal human T cells accumulate substantial amounts of deoxyadenosine 5'-triphosphate when incubated in medium supplemented with deoxyadenosine and an adenosine deaminase inhibitor. Seto et al. (1985) J. Clin. Invest., 75:377-383; and Kefford et al. (1982) Cancer Res., 42:324-330.
Deoxyadenosine 5-triphosphate causes the secretion of DNA single-strand breaks in resting, normal T cells, presumably by inhibiting DNA polymerase-alpha. Seto et al. (1985) J. Clin. Invest., 75:377-383; Seto et al. (1986) J. Immunol., 136:2839-2843. Such DNA breaks trigger a programmed "suicide" response in the T cells that is associated with a lethal depletion of NAD and ATP pools, during exhaustive poly(ADP-ribose) synthesis.
One report suggests that 2',3'-dideoxynucleoside 5'-triphosphates are not substrates for DNA polymerase-alpha Edenberg et al. (1978) J. Biol. Chem., 253:3273-3280. Preliminary work from our own laboratories indicates that such compounds should not inhibit DNA repair in human normal peripheral blood lymphocytes.
Work directed toward the inhibition of growth of human malignant T cell lines in our own laboratories examined the resistance of adenine derivatives to deamination by adenosine deaminase and the toxicity of resistant derivatives toward human T and B lymphoblastoid cell lines. The most potent agent, on a molar basis, was found to be 2-chloro-2'-deoxyadenosine.
That compound was able to inhibit growth of human malignant T cell lines at concentrations of 0.001-0.03 micromolar (uM). Cell lines derived from solid tissues, e.g., Hela cells and normal fibroblasts, were found to be about 100-times more resistant to that adenine derivative. 2-Chloro-2'-deoxyadenosine can be toxic toward fresh T cells, but has no discernable effects on mature granulocytes. Carson et al. (1983) Blood, 62:737-743.
2-Chloro-2'-deoxyadenosine is phosphorylated by non-dividing (normal) human peripheral blood lymphocytes and is converted to the 5'-triphosphate. This adenine derivative is not catabolized significantly by intact human cells or cell extracts, and is phosphorylated efficiently by T lymphocytes. Carson et al. (1980) Proc. Natl. Acad. Sci. USA, 77:6865-6869.
Phase 1 studies on humans showed infusion of increasing doses of that 2-chloro-2'-deoxyadenosine [0.1-0.5 milligrams per kilogram of body weight per day (mg/kg/day)] yielded increasing plasma concentrations of the drug [10-50 nanomolar (nM)]. Those infusions indicated that that drug was well tolerated and did not induce nausea, vomiting or fever. The dose-limiting toxicity was bone marrow suppression, which usually occurred at doses greater than about 0.2 mg/kg/day or at plasma levels of greater than about 20 nM.
Other studies, Montgomery et al. (1960) J. Am. Chem. Soc., 82:463-468, indicated that 2-fluoroadenosine exhibits a relatively high degree of cytotoxicity. Those workers reported that C57 black mice implanted with Adenocarcinoma 755 (Ad755) could tolerate only about 1 milligram per kilogram of body weight. 2-Fluoroadenosine was found to be inactive at that level against Ad755 as well as leukemia L1210 and the Erlich ascites tumor.
In work recently published [Herdewijn et al. (1987) J. Med. Chem., 30:1270-1278], the activity against HLV and the toxicity in T cells are reported for a number of nucleosides. Among the compounds discussed were ddA, 3'-fluoro-ddA, and 3'-azido-ddA.
Concentrations providing a 50 percent protection level of HIV-infected ATH8 cells against the cytopathic effect of HIV were reported to be 2.7, 8 and 4.8 micromolar, respectively. Doses required to reduce the viability of normal, uninfected ATH8 cells by 50 percent were determined to be more than 500, more than 250 and 27 micromolar, respectively.
An adenine derivative that is: (a) capable of traversing the cellular membrane from the medium, (b) capable of being phosphorylated to the 5'-triphosphate once inside a T cell, (c) substantially free from deamination, (d) a non-inhibitor for DNA polymerase-alpha, (e) capable of inhibiting retroviral replication such as by terminating a growing RNA or DNA chain, and (f) relatively non-toxic to the retrovirally-infected cells and generally to the host animal might be an improved chemotherapeutic agent against a retrovirally-induced disease such as AIDS. The following disclosure describes a relatively small group of such compounds.